Processing by Endoplasmic Reticulum Mannosidases Partitions a Secretion-impaired Glycoprotein into Distinct Disposal Pathways

Christopher M. Cabral, Priya Choudhury, Yan Liu, Richard N. Sifers
2000 Journal of Biological Chemistry  
In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded ␣ 1 -antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from
more » ... lnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861-5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed. In the endoplasmic reticulum (ER), 1 an assortment of molecular chaperones and folding enzymes facilitate the conformational maturation of newly synthesized polypeptides destined for deployment to the cell surface as biologically active proteins (1). In recent years, a picture has emerged that describes how asparagine-linked glycosylation, in combination with several independently acting enzymes, facilitates glycoprotein folding (for reviews, see Refs. 2 and 3). In a widely accepted model (4) , the partial deglucosylation of asparagine-linked Glc 3 Man 9 -GlcNAc 2 induces cotranslational physical interaction between glycoproteins and members of a small family of lectins, each of which recognizes the monoglucosylated glycan as ligand (2) . Dissociation of the complex coincides with the removal of glucose by glucosidase II (5). In the absence of conformational maturation, UDP-glucose:glycoprotein glucosyltransferase (UGTR) functions as a folding sensor (6) that recognizes struc-tural determinants common to nonnative glycoprotein structure (7, 8). Reglucosylation of asparagine-linked oligosaccharides induces the reassembly of folding intermediates with calnexin (4). As such, reversible glucosylation hinders premature exit from the ER (2, 4, 9) until correctly folded molecules that are no longer substrates for UGTR are released from the lectin-mediated retention cycle (4). As a rule, failure to attain conformational maturation following biosynthesis results in the selective elimination of misfolded polypeptides and unassembled protein complexes by a relatively stringent mechanism of conformation-based quality control (10, 11). Molecular characterization of primary and secondary disposal systems, plus the identification of the full repertoire of quality control machinery, is currently under intense investigation (for a review, see Ref. 11). Because the efficiency of modification by UGTR and glucosidase II is sensitive to the number of mannose units within the asparaginelinked oligosaccharide (6, 13), it is possible that oligosaccharide processing represents the meeting point between protein folding and quality control pathways. For this reason, recent work has focused toward elucidating the potential role of processing mannosidases in glycoprotein quality control (9, 14, 15) . In addition to its fundamental importance in normal cell physiology (16), the process of quality control in the early secretory pathway has been implicated as a key factor in the molecular pathogenesis associated with several human disorders (17, 18) . To this end, a major physiologic role for the monomeric secretory glycoprotein ␣ 1 -antitrypsin (AAT) is to prevent the destruction of lung elastin. The hindered secretion and disposal of allelic variants from liver hepatocytes, the predominant site of biosynthesis (19), can lead to plasma AAT deficiency. A severe deficiency of the plasma protein is known to function as a heritable risk factor for the development of chronic obstructive lung disease (for reviews, see Refs. 20 and 21). Gene expression studies performed in stably transfected murine hepatoma cells have allowed for the characterization of AAT quality control mechanisms in a physiologically relevant model system (9, (22) (23) (24) (25) . The truncation of carboxyl-terminal amino acids in genetic variant PI QO Hong Kong (null(Hong Kong)) (22) precludes conformational maturation following biosynthesis, resulting in its lectin-mediated intracellular retention prior to disposal (9). Recently, we (26) proposed a model of quality control in which the removal of a single terminal ␣1,2linked mannose unit from multiple asparagine-linked oligosaccharides abrogates the physical dissociation of null(Hong Kong) from the ER lectin calnexin (27) , leading to its selective degradation by the cytosolic proteasome (28). In this process of "molecular capture," the attenuated removal of glucose from asparagine-linked oligosaccharides functions as the underlying mechanism by which the misfolded glycoprotein is selected for degradation. In the present study, stably transfected hepatoma cells were
doi:10.1074/jbc.m910172199 pmid:10827201 fatcat:ddntvvunb5athkj2od6vut6cgm